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In this article we will discuss about the markers for chloroplast transformation.
Generally, dominant antibiotic resistant genes are used in plastid transformation. Introduction of antibiotic resistant gene in the plastid genome facilitated the maintenance of high number of plastid genome copies in a cell. Chloroplast transformation invariably and unavoidably produces chimeric tissues, which requires identification of transplastomic cells in order to regenerate plants.
Thus, antibiotic resistant genes are indispensible in plastid transformation. Specific choice for spectinomycin is due to its prokaryotic translational inhibitor character and has meagre impact over plant cells.
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Therefore, spectinomycin is the most prominent selectable marker gene for chloroplast transformation. In addition, neomycin phosphotransferase (NPt 11) gene which confer kanamycin resistance may also be employed for chloroplast transformation.
Selection of transplastomic lines requires the drug, which inhibit chloroplast accumulation and shoot formation on plant culture media. When chloroplast genome were transformed by antibiotic resistant genes like aad A and neo, which confer resistant to the drug and facilitated the selection of transplastomic lines.
Morphologically, these are identified by ability to form green shoots on bleached wild type leaf section. Plastid transformation takes place in one or few genome copies within a single plant cell, resulting in cells that contain a mix of transformed and wild type chloroplast genome. These cells are referred as heteroplastomic.
These are again categorised into interplastidic and intraplastidic. Interplastidic meteroplastomic chloroplast contains wild type genome and transferred genomes. Intraplastidic heteroplasm is where wild type and transformed genomes are located within the same chloroplast.
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Stable transplastomic lines requires cultivation of cells on a selection media, in which cells undergoes spontaneous division for atleast 1&-17 times. During these periods, wild type and transformed plastids and plastid genome copies gradually sort out.
In this process, resultant chimeric plants consist of wild type and transgenic cells. Since, both the wild type and transplastomic sector looks green due to phenotypic masking by the transgenic tissue; it requires second cycle of plant regeneration on selection media.
In order to faciliate screening by visual marker, homoplastomic clones are screened by visually identifying transformed vectors. The visual marker are generally a green fluorescent protein (GFP), derived from Aequorea victoria, which facilitate direct imaging of the fluorescence gene product in cells without causing cumbersome histochemical staining methods.
Its chromophores forms auto catalytically in the presence of oxygen and fluorescence green when absorbing blue or ultraviolet (uv) light. Recently, gfp gene was modified for expression in the plant by removing cryptic intron and introducing mutation to enhance brightness when observed.
Green fluorescence protein was used for protein targetting to plastid from nuclear genes. Successful injection of the gfp gene into the attached leaves of tobacco and Vicia faba by femtosyringe resulted in the production of green fluorescent protein (GFP) within chloroplast.
The use of new fluorescent antibiotic resistant enzyme conferring resistant to spectinomycin/streptomycin (FLAR-S) to identify plastid transformation. The FLARE-S was obtained by fusing an enzyme amino glycosideadenyl transferase with the green fluorescent protein. This combined marker unit facilitate segregation of individual transformed and wild type plastid (Fig. 19.4). This technique was successfully applied in transformation of tobacco and rice chloroplast.
